Methods and compositions for detection and analysis of polynucleotides using light harvesting multichromophores

ABSTRACT

Methods, compositions and articles of manufacture for assaying a sample for a target polynucleotide are provided. A sample suspected of containing the target polynucleotide is contacted with a polycationic multichromophore and a sensor polynucleotide complementary to the target polynucleotide. The sensor polynucleotide comprises a signaling chromophore to receive energy from the excited multichromophore and increase emission in the presence of the target polynucleotide. The methods can be used in multiplex form. Kits comprising reagents for performing such methods are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/854,365, filed Sep. 12, 2007, now U.S. Pat. No. 7,629,448 issued Dec.8, 2009, which claims the benefit of U.S. patent application Ser. No.10/648,945 filed Aug. 26, 2003, now U.S. Pat. No. 7,270,956, issued Sep.18, 2007, and of U.S. Provisional Application No. 60/406,266, filed Aug.26, 2002. The aforementioned applications are hereby incorporated hereinby reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under grant numberDMR-0097611 awarded by the National Science Foundation. The Governmenthas certain rights in this invention.

TECHNICAL FIELD

This invention relates to methods, articles and compositions for thedetection and analysis of polynucleotides in a sample.

BACKGROUND OF THE INVENTION

Methods permitting DNA sequence detection in real time and with highsensitivity are of great scientific and economic interest.^(1,2,3) Theirapplications include medical diagnostics, identification of geneticmutations, gene delivery monitoring and specific genomic techniques.⁴Cationic organic dyes, such as ethidium bromide and thiazole orange,emit when intercalated into the grooves of double strand DNA (dsDNA),and serve as direct DNA hybridization probes, but lack sequencespecificity.^(5,6) Energy/electron transfer chromophore pairs for strandspecific assays exist, but require chemical labeling of two nucleicacids, or dual modification of the same altered strand (for example,molecular beacons).^(7,8) Difficulties in labeling two DNA sites resultin low yields, high costs and singly labeled impurities, which lowerdetection sensitivity.⁹

There is a need in the art for methods of detecting and analyzingparticular polynucleotides in a sample, and for compositions andarticles of manufacture useful in such methods.

SUMMARY OF THE INVENTION

Methods, compositions and articles of manufacture for detecting andassaying a target polynucleotide in a sample are provided.

A sample suspected of containing the target polynucleotide is contactedwith a polycationic multichromophore and a sensor polynucleotidecomplementary to the target polynucleotide. The sensor polynucleotidecomprises an anionic backbone, such as a typical sugar phosphatebackbone, and is conjugated to a signaling chromophore. In the presenceof target polynucleotide in the sample, the signaling chromophore canacquire energy more efficiently from the excited polycationicmultichromophore and emit increased amounts of light or signal which canbe detected. The target polynucleotide can be analyzed as it occurs inthe sample, or can be amplified prior to or in conjunction withanalysis.

Although the anionic sensor can associate with the cationicmultichromophore in the absence of target, a surprising increase insignal has been found to occur upon binding of the target polynucleotideto form a double-stranded complex relative to the signal produced from amixture of non-complementary sequences. This increase in signal can beexploited for use in detection methods for assaying for a targetpolynucleotide in a sample.

Solutions are provided comprising reagents useful for performing themethods of the invention, as are kits containing such reagents. Alsoprovided are sensing or detection complexes formed from themultichromophore and sensor polynucleotide, and signaling complexesfurther comprising the target polynucleotide. The methods can be used inmultiplex settings where a plurality of different sensor polynucleotidesare used to assay for a plurality of different target polynucleotides.The methods can optionally be performed on a surface, for example usinga surface-associated polycationic multichromophore; the surface can be asensor. The methods can also be provided in homogeneous formats. Themethods and articles described herein can be used as alternatives toother techniques for detecting polynucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the absorption (a (dotted line) and c (dashed line)) andemission (b (squares) and d (circles)) spectra of polymer 1 and afluorescein-conjugated sensor polynucleotide. Excitation was done at 380and 480 nm for polymer 1 and sensor polynucleotide, respectively. Theenergy transfer complex of polymer 1 and sensor polynucleotide excitedat 380 nm is also shown in black (e, solid line).

FIG. 2 presents the emission spectra of the sensor system containinghybridized (solid line) and non-hybridized (dotted line) sensorpolynucleotides in 10 mmol Sodium Citrate and 100 mmol sodium chloridebuffer at pH=8. Spectra are normalized relative to the emission ofpolymer 1.

FIG. 3 depicts the emission spectra of the sensor system containinghybridized (solid line) and non-hybridized (dotted line) sensorpolynucleotides resulting from excitation of the multichromophore(polymer 1) and energy transfer to the sensor polynucleotide followed bysubsequent energy transfer to a polynucleotide specific dye (EthidiumBromide). Measurements are in potassium phosphate-sodium hydroxidebuffer solution (50 mM, pH=7.40). See Example 4.

FIG. 4 depicts the amplified emission spectra of apolynucleotide-specific dye (Ethidium Bromide, EB) in the sensor systemcontaining a hybridized (solid line) sensor polynucleotide. Theamplified emission signal is a result of excitation of themultichromophore (polymer 1) and energy transfer to a sensorpolynucleotide followed by subsequent energy transfer to EB. Direct EBemission in double stranded DNA (dotted line) and the emission resultingfrom sensor polynucleotide excitation followed by energy transfer to EB(dashed line) is shown to demonstrate the increased signal provided bythe polycationic multichromophore. Measurements are in potassiumphosphate-sodium hydroxide buffer solution (50 mM, pH=7.40). See Example5.

FIG. 5 presents the emission spectra of EB upon excitation of polymer 1in the presence of EB and both hybridized (solid line) andnon-hybridized (dotted line) sensor polynucleotides in potassiumphosphate-sodium hydroxide buffer solution (50 mM, pH=7.40). See Example6. The emission is a result of energy transfer from the polycationicmultichromophore to the intercalated EB only in the case of thehybridized, double stranded DNA.

DETAILED DESCRIPTION OF THE INVENTION

Present technologies for DNA and RNA sensors (including “gene-chips” and“DNA-chips”) depend on the covalent attachment of fluorescent tags(lumophores) to single strands of DNA, relying on labeling of the sampleor a sample component, with unavoidable problems resulting fromvariations in the efficiency of the labeling reaction from sample tosample, requiring complex cross-calibrations. Other systems rely onmultiply labeled probes (e.g., molecular beacons, Taqman®, Scorpion®probes, Eclipse® probes), requiring the multiple attachment oflumophores and quenchers to precisely engineered sequences.

The method of the invention comprises contacting a sample with anaqueous solution comprising at least two components; (a) a lightharvesting, polycationic, luminescent multichromophore system such as,for example, a conjugated polymer, semiconductor quantum dot ordendritic structure that is water soluble, and (b) a sensorpolynucleotide conjugated to a luminescent signaling chromophore(referred to as “Oligo-C*”). The emission of light with wavelengthcharacteristic of the signaling chromophore indicates the presence insolution of a target polynucleotide with a base sequence complementaryto that of the sensor polynucleotide. By using multiple sensorpolynucleotides, each with a different base sequence and a differentsignaling chromophore (Oligo₁-C₁*, Oligo₂-C₂*, etc), multiplepolynucleotides each with specific base sequences can be independentlydetected. A third component such as a polynucleotide-specific dye may beintroduced to improve selectivity by further transferring energy fromthe sensor polynucleotide to the polynucleotide-specific dye.

The light harvesting chromophore and the signaling chromophore (C*) arechosen so that the absorption bands of the two species have minimaloverlap and so that the luminescent emission spectra of the two speciesare at different wavelengths. When prepared in aqueous solution, thelight harvesting and luminescent multi-chromophore systems arepositively charged (for example positively charged conjugatedpolyelectrolytes). The proximity between a negatively charged sensorpolynucleotide and a positively charged light harvesting and luminescentmulti-chromophore system is ensured by electrostatic attraction. Whenexposed to incident radiation with wavelength in the absorption band ofthe light harvesting chromophore, there is emission from the signalingchromophore, for example via the Förster energy transfer mechanism. Uponaddition of a target polynucleotide such as a single stranded DNA(ssDNA) with a base sequence that is complementary to the sequence ofthe sensor polynucleotide, the ssDNA hybridizes with the sensorpolynucleotide resulting in an increase in negative charge density alongthe DNA strand.^(10,11) Under these circumstances, and consideringstrictly electrostatic forces, the interaction between the sensorpolynucleotide and the positively charged light harvestingmulti-chromophore system will be favorable, leading to efficient energytransfer and intense emission from the signaling chromophore. When ssDNAwith a base sequence that is not complementary to that of the sensorpolynucleotide is added, base pair hybridization does not take place.Electrostatic complexation between a light harvesting multi-chromophoresystem and a sensor polynucleotide is screened by competition with thenon-complementary ssDNA. Thus, the average distance between the lightharvesting multi-chromophore system and the Oligo-C* is larger in thepresence of a noncomplementary sequence, resulting in less effectiveenergy transfer to the signaling chromophore. The emission of thesignaling Oligo-C* is stronger when in the presence of its complementarytarget than when a non-complementary strand is present. The overallscheme provides a sensor for the presence of a target polynucleotidewith a specific base sequence in the test solution. By using multiplesensor polynucleotides, each with a different base sequence and adifferent signaling chromophore, multiple targets can be separatelydetected.

In addition to the described method, the invention provides apredominantly aqueous solution comprising at least two components; (a) acationic multichromophore, and (b) a “sensor polynucleotide” (Oligo-C*)comprising an anionic polynucleotide conjugated to a signalingchromophore.

As demonstrated in the Examples, the optical amplification provided by awater soluble multichromophore such as a conjugated polymer can be usedto detect polynucleotide hybridization to a sensor polynucleotide. Theamplification can be enhanced by using higher molecular weight watersoluble conjugated polymers or other structures as the polycationicmultichromophore as described herein. The invention can be provided in ahomogeneous format that utilizes the ease of fluorescence detectionmethods. The invention can be used to detect amplified targetpolynucleotides or, because of the large signal amplification, as astand alone assay, without need for polynucleotide amplification.

Unlike gene-chip technology, the present invention does not necessarilyrequire labeling of each sample to be analyzed by covalent coupling oflumophores or chromophores to the polynucleotides contained in orderived from the sample prior to analysis. Those coupling methods haveinherent difficulties in reproducibility of coupling efficiency andresult in the need for cross-calibration from sample to sample.

The inventions described herein are useful for any assay in which asample can be interrogated regarding a target polynucleotide. Typicalassays involve determining the presence of the target polynucleotide inthe sample or its relative amount, or the assays may be quantitative orsemi-quantitative.

The methods of the invention can all be performed in multiplex formats.A plurality of different sensor polynucleotides can be used to detectcorresponding different target polynucleotides in a sample through theuse of different signaling chromophores conjugated to the respectivesensor polynucleotides. Multiplex methods are provided employing 2, 3,4, 5, 10, 15, 20, 25, 50, 100, 200, 400 or more different sensorpolynucleotides which can be used simultaneously to assay forcorresponding different target polynucleotides.

The methods can be performed on a substrate, as well as in solution,although the solution format is expected to be more rapid due todiffusion issues. Thus the assay can be performed, for example, in anarray format on a substrate, which can be a sensor. This can be achievedby anchoring or otherwise incorporating an assay component onto thesubstrate, for example the sensor polynucleotide, the polycationicmultichromophore, or both. These substrates may be surfaces of glass,silicon, paper, plastic, or the surfaces of optoelectronicsemiconductors (such as, but not confined to, indium-doped galliumnitride or polymeric polyanilines, etc.) employed as optoelectronictransducers. The location of a given sensor polynucleotide may be knownor determinable in an array format, and the array format may bemicroaddressable or nanoaddressable. In one variation, one or moresamples, which may contain an amplification product, can be attached tothe substrate, and the substrate can be contacted with one or morelabeled sensor polynucleotides and the polycationic multichromophore.

Before the present invention is described in further detail, it is to beunderstood that this invention is not limited to the particularmethodology, devices, solutions or apparatuses described, as suchmethods, devices, solutions or apparatuses can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention.

Use of the singular forms “a,” “an,” and “the” include plural referencesunless the context clearly dictates otherwise. Thus, for example,reference to “a target polynucleotide” includes a plurality of targetpolynucleotides, reference to “a signaling chromophore” includes aplurality of such chromophores, reference to “a sensor polynucleotide”includes a plurality of sensor polynucleotides, and the like.Additionally, use of specific plural references, such as “two,” “three,”etc., read on larger numbers of the same subject less the contextclearly dictates otherwise.

Terms such as “connected,” “attached,” and “linked” are usedinterchangeably herein and encompass direct as well as indirectconnection, attachment, linkage or conjugation unless the contextclearly dictates otherwise. Where a range of values is recited, it is tobe understood that each intervening integer value, and each fractionthereof, between the recited upper and lower limits of that range isalso specifically disclosed, along with each subrange between suchvalues. The upper and lower limits of any range can independently beincluded in or excluded from the range, and each range where either,neither or both limits are included is also encompassed within theinvention. Where a value being discussed has inherent limits, forexample where a component can be present at a concentration of from 0 to100%, or where the pH of an aqueous solution can range from 1 to 14,those inherent limits are specifically disclosed. Where a value isexplicitly recited, it is to be understood that values which are aboutthe same quantity or amount as the recited value are also within thescope of the invention, as are ranges based thereon. Where a combinationis disclosed, each subcombination of the elements of that combination isalso specifically disclosed and is within the scope of the invention.Conversely, where different elements or groups of elements aredisclosed, combinations thereof are also disclosed. Where any element ofan invention is disclosed as having a plurality of alternatives,examples of that invention in which each alternative is excluded singlyor in any combination with the other alternatives are also herebydisclosed; more than one element of an invention can have suchexclusions, and all combinations of elements having such exclusions arehereby disclosed.

Unless defined otherwise or the context clearly dictates otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. Although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the invention, the preferred methods and materials are nowdescribed.

All publications mentioned herein are hereby incorporated by referencefor the purpose of disclosing and describing the particular materialsand methodologies for which the reference was cited. The publicationsdiscussed herein are provided solely for their disclosure prior to thefiling date of the present application. Nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue of prior invention.

Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and“nucleic acid molecule” are used interchangeably herein to refer to apolymeric form of nucleotides of any length, and may compriseribonucleotides, deoxyribonucleotides, analogs thereof, or mixturesthereof. These terms refer only to the primary structure of themolecule. Thus, the terms includes triple-, double- and single-strandeddeoxyribonucleic acid (“DNA”), as well as triple-, double- andsingle-stranded ribonucleic acid (“RNA”). It also includes modified, forexample by alkylation, and/or by capping, and unmodified forms of thepolynucleotide.

Whether modified or unmodified, the sensor polynucleotide is anionic andcan interact with the cationic multichromophore in the absence of targetpolynucleotide. The target polynucleotide can in principle be charged oruncharged, although typically it is expected to be anionic, for exampleRNA or DNA.

More particularly, the terms “polynucleotide,” “oligonucleotide,”“nucleic acid” and “nucleic acid molecule” includepolydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA,and mRNA, whether spliced or unspliced, any other type of polynucleotidewhich is an N- or C-glycoside of a purine or pyrimidine base, and otherpolymers containing a phosphate or other polyanionic backbone, and othersynthetic sequence-specific nucleic acid polymers providing that thepolymers contain nucleobases in a configuration which allows for basepairing and base stacking, such as is found in DNA and RNA. There is nointended distinction in length between the terms “polynucleotide,”“oligonucleotide,” “nucleic acid” and “nucleic acid molecule,” and theseterms are used interchangeably herein. These terms refer only to theprimary structure of the molecule. Thus, these terms include, forexample, 3′-deoxy-2′,5′-DNA, oligodeoxyribonucleotide N3′ P5′phosphoramidates, 2′-O-alkyl-substituted RNA, double- andsingle-stranded DNA, as well as double- and single-stranded RNA, andhybrids thereof including for example hybrids between DNA and RNA, andalso include known types of modifications, for example, labels,alkylation, “caps,” substitution of one or more of the nucleotides withan analog, internucleotide modifications such as, for example, thosewith negatively charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (including enzymes (e.g. nucleases), toxins,antibodies, signal peptides, poly-L-lysine, etc.), those withintercalators (e.g., acridine, psoralen, etc.), those containingchelates (of, e.g., metals, radioactive metals, boron, oxidative metals,etc.), those containing alkylators, those with modified linkages (e.g.,alpha anomeric nucleic acids, etc.), as well as unmodified forms of thepolynucleotide or oligonucleotide.

It will be appreciated that, as used herein, the terms “nucleoside” and“nucleotide” will include those moieties which contain not only theknown purine and pyrimidine bases, but also other heterocyclic baseswhich have been modified. Such modifications include methylated purinesor pyrimidines, acylated purines or pyrimidines, or other heterocycles.Modified nucleosides or nucleotides can also include modifications onthe sugar moiety, e.g., wherein one or more of the hydroxyl groups arereplaced with halogen, aliphatic groups, or are functionalized asethers, amines, or the like. The term “nucleotidic unit” is intended toencompass nucleosides and nucleotides.

Furthermore, modifications to nucleotidic units include rearranging,appending, substituting for or otherwise altering functional groups onthe purine or pyrimidine base which form hydrogen bonds to a respectivecomplementary pyrimidine or purine. The resultant modified nucleotidicunit optionally may form a base pair with other such modifiednucleotidic units but not with A, T, C, G or U. Abasic sites may beincorporated which do not prevent the function of the polynucleotide;preferably the polynucleotide does not comprise abasic sites. Some orall of the residues in the polynucleotide can optionally be modified inone or more ways.

Standard A-T and G-C base pairs form under conditions which allow theformation of hydrogen bonds between the N3-H and C4-oxy of thymidine andthe N1 and C6-NH2, respectively, of adenosine and between the C2-oxy, N3and C4-NH2, of cytidine and the C2-NH2, N′-H and C6-oxy, respectively,of guanosine. Thus, for example, guanosine(2-amino-6-oxy-9-β-D-ribofuranosyl-purine) may be modified to formisoguanosine (2-oxy-6-amino-9-β-D-ribofuranosyl-purine). Suchmodification results in a nucleoside base which will no longereffectively form a standard base pair with cytosine. However,modification of cytosine (1-β-D-ribofuranosyl-2-oxy-4-amino-pyrimidine)to form isocytosine (1-β-D-ribofuranosyl-2-amino-4-oxy-pyrimidine)results in a modified nucleotide which will not effectively base pairwith guanosine but will form a base pair with isoguanosine. Isocytosineis available from Sigma Chemical Co. (St. Louis, Mo.); isocytidine maybe prepared by the method described by Switzer et al. (1993)Biochemistry 32:10489-10496 and references cited therein;2′-deoxy-5-methyl-isocytidine may be prepared by the method of Tor etal. (1993) J. Am. Chem. Soc. 115:4461-4467 and references cited therein;and isoguanine nucleotides may be prepared using the method described bySwitzer et al. (1993), supra, and Mantsch et al. (1993) Biochem.14:5593-5601, or by the method described in U.S. Pat. No. 5,780,610 toCollins et al. Other nonnatural base pairs may be synthesized by themethod described in Piccirilli et al. (1990) Nature 343:33-37 for thesynthesis of 2,6-diaminopyrimidine and its complement(1-methylpyrazolo-[4,3]pyrimidine-5,7-(4H,6H)-dione). Other suchmodified nucleotidic units which form unique base pairs are known, suchas those described in Leach et al. (1992) J. Am. Chem. Soc.114:3675-3683 and Switzer et al., supra.

“Preferential binding” or “preferential hybridization” refers to theincreased propensity of one polynucleotide or PNA to bind to itscomplement in a sample as compared to a noncomplementary polymer in thesample.

Hybridization conditions will typically include salt concentrations ofless than about 1M, more usually less than about 500 mM and preferablyless than about 200 mM. In the case of hybridization between a peptidenucleic acid and a polynucleotide, the hybridization can be done insolutions containing little or no salt. Hybridization temperatures canbe as low as 5° C., but are typically greater than 22° C., moretypically greater than about 30° C., and preferably in excess of about37° C. Longer fragments may require higher hybridization temperaturesfor specific hybridization. Other factors may affect the stringency ofhybridization, including base composition and length of thecomplementary strands, presence of organic solvents and extent of basemismatching, and the combination of parameters used is more importantthan the absolute measure of any one alone. Suitable hybridizationconditions for a given assay format can be determined by one of skill inthe art; nonlimiting parameters which may be adjusted includeconcentrations of assay components, salts used and their concentration,ionic strength, temperature, buffer type and concentration, solution pH,presence and concentration of blocking reagents to decrease backgroundbinding such as repeat sequences or blocking protein solutions,detergent type(s) and concentrations, molecules such as polymers whichincrease the relative concentration of the polynucleotides, metal ion(s)and their concentration(s), chelator(s) and their concentrations, andother conditions known in the art.

“Multiplexing” herein refers to an assay or other analytical method inwhich multiple analytes can be assayed simultaneously.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where the event or circumstance occurs and instances in whichit does not.

The Sample

The portion of the sample comprising or suspected of comprising thetarget polynucleotide can be any source of biological material whichcomprises polynucleotides that can be obtained from a living organismdirectly or indirectly, including cells, tissue or fluid, and thedeposits left by that organism, including viruses, mycoplasma, andfossils. The sample may comprise a target polynucleotide preparedthrough synthetic means, in whole or in part. Typically, the sample isobtained as or dispersed in a predominantly aqueous medium. Nonlimitingexamples of the sample include blood, urine, semen, milk, sputum, mucus,a buccal swab, a vaginal swab, a rectal swab, an aspirate, a needlebiopsy, a section of tissue obtained for example by surgery or autopsy,plasma, serum, spinal fluid, lymph fluid, the external secretions of theskin, respiratory, intestinal, and genitourinary tracts, tears, saliva,tumors, organs, samples of in vitro cell culture constituents (includingbut not limited to conditioned medium resulting from the growth of cellsin cell culture medium, putatively virally infected cells, recombinantcells, and cell components), and a recombinant library comprisingpolynucleotide sequences. The sample may be presented on a substrate asdescribed herein. The substrate may be a slide comprising the sample,such as is used in fluorescence in situ hybridization (FISH).

The sample can be a positive control sample which is known to containthe target polynucleotide or a surrogate thereof. A negative controlsample can also be used which, although not expected to contain thetarget polynucleotide, is suspected of containing it (via contaminationof one or more of the reagents) or another component capable ofproducing a false positive, and is tested in order to confirm the lackof contamination by the target polynucleotide of the reagents used in agiven assay, as well as to determine whether a given set of assayconditions produces false positives (a positive signal even in theabsence of target polynucleotide in the sample).

The sample can be diluted, dissolved, suspended, extracted or otherwisetreated to solubilize and/or purify any target polynucleotide present orto render it accessible to reagents which are used in an amplificationscheme or to detection reagents. Where the sample contains cells, thecells can be lysed or permeabilized to release the polynucleotideswithin the cells. One step permeabilization buffers can be used to lysecells which allow further steps to be performed directly after lysis,for example a polymerase chain reaction.

The Target Polynucleotide and Amplification Products Produced Therefrom

The target polynucleotide can be single-stranded, double-stranded, orhigher order, and can be linear or circular. Exemplary single-strandedtarget polynucleotides include mRNA, rRNA, tRNA, hnRNA, ssRNA or ssDNAviral genomes, although these polynucleotides may contain internallycomplementary sequences and significant secondary structure. Exemplarydouble-stranded target polynucleotides include genomic DNA,mitochondrial DNA, chloroplast DNA, dsRNA or dsDNA viral genomes,plasmids, phage, and viroids. The target polynucleotide can be preparedsynthetically or purified from a biological source. The targetpolynucleotide may be purified to remove or diminish one or moreundesired components of the sample or to concentrate the targetpolynucleotide. Conversely, where the target polynucleotide is tooconcentrated for the particular assay, the target polynucleotide may bediluted.

Following sample collection and optional nucleic acid extraction, thenucleic acid portion of the sample comprising the target polynucleotidecan be subjected to one or more preparative reactions. These preparativereactions can include in vitro transcription (IVT), labeling,fragmentation, amplification and other reactions. mRNA can first betreated with reverse transcriptase and a primer to create cDNA prior todetection and/or amplification; this can be done in vitro with purifiedmRNA or in situ, e.g. in cells or tissues affixed to a slide. Nucleicacid amplification increases the copy number of sequences of interestsuch as the target polynucleotide. A variety of amplification methodsare suitable for use; nonlimiting examples of suitable amplificationreactions include the polymerase chain reaction method (PCR), the ligasechain reaction (LCR), self sustained sequence replication (3SR), nucleicacid sequence-based amplification (NASBA), the use of Q Beta replicase,reverse transcription, nick translation, and the like.

Where the target polynucleotide is single-stranded, the first cycle ofamplification forms a primer extension product complementary to thetarget polynucleotide. If the target polynucleotide is single-strandedRNA, a polymerase with reverse transcriptase activity is used in thefirst amplification to reverse transcribe the RNA to DNA, and additionalamplification cycles can be performed to copy the primer extensionproducts. The primers for a PCR must, of course, be designed tohybridize to regions in their corresponding template that will producean amplifiable segment; thus, each primer must hybridize so that its 3′nucleotide is paired to a nucleotide in its complementary templatestrand that is located 3′ from the 3′ nucleotide of the primer used toreplicate that complementary template strand in the PCR.

The target polynucleotide is typically amplified by contacting one ormore strands of the target polynucleotide with a primer and a polymerasehaving suitable activity to extend the primer and copy the targetpolynucleotide to produce a full-length complementary polynucleotide ora smaller portion thereof. Any enzyme having a polymerase activity whichcan copy the target polynucleotide can be used, including DNApolymerases, RNA polymerases, reverse transcriptases, enzymes havingmore than one type of polymerase activity, and the enzyme can bethermolabile or thermostable. Mixtures of enzymes can also be used.Exemplary enzymes include: DNA polymerases such as DNA Polymerase I(“Pol I”), the Klenow fragment of Pol I, T4, T7, Sequenase® T7,Sequenase® Version 2.0 T7, Tub, Taq, Tth, Pfx, Pfu, Tsp, Tfl, Tli andPyrococcus sp GB-D DNA polymerases; RNA polymerases such as E. coli,SP6, T3 and T7 RNA polymerases; and reverse transcriptases such as AMV,M-MuLV, MMLV, RNAse H³¹ MMLV (SuperScript®), SuperScript® II,ThermoScript®, HIV-1, and RAV2 reverse transcriptases. All of theseenzymes are commercially available. Exemplary polymerases with multiplespecificities include RAV2 and Tli (exo-) polymerases. Exemplarythermostable polymerases include Tub, Taq, Tth, Pfx, Pfu, Tsp, Tfl, Tliand Pyrococcus sp. GB-D DNA polymerases.

Suitable reaction conditions are chosen to permit amplification of thetarget polynucleotide, including pH, buffer, ionic strength, presenceand concentration of one or more salts, presence and concentration ofreactants and cofactors such as nucleotides and magnesium and/or othermetal ions (e.g., manganese), optional cosolvents, temperature, thermalcycling profile for amplification schemes comprising a polymerase chainreaction, and may depend in part on the polymerase being used as well asthe nature of the sample. Cosolvents include formamide (typically atfrom about 2 to about 10%), glycerol (typically at from about 5 to about10%), and DMSO (typically at from about 0.9 to about 10%). Techniquesmay be used in the amplification scheme in order to minimize theproduction of false positives or artifacts produced duringamplification. These include “touchdown” PCR, hot-start techniques, useof nested primers, or designing PCR primers so that they form stem-loopstructures in the event of primer-dimer formation and thus are notamplified. Techniques to accelerate PCR can be used, for examplecentrifugal PCR, which allows for greater convection within the sample,and comprising infrared heating steps for rapid heating and cooling ofthe sample. One or more cycles of amplification can be performed. Anexcess of one primer can be used to produce an excess of one primerextension product during PCR; preferably, the primer extension productproduced in excess is the amplification product to be detected. Aplurality of different primers may be used to amplify different targetpolynucleotides or different regions of a particular targetpolynucleotide within the sample.

Amplified target polynucleotides may be subjected to post amplificationtreatments. For example, in some cases, it may be desirable to fragmentthe target polynucleotide prior to hybridization in order to providesegments which are more readily accessible. Fragmentation of the nucleicacids can be carried out by any method producing fragments of a sizeuseful in the assay being performed; suitable physical, chemical andenzymatic methods are known in the art.

An amplification reaction can be performed under conditions which allowthe sensor polynucleotide to hybridize to the amplification productduring at least part of an amplification cycle. When the assay isperformed in this manner, real-time detection of this hybridizationevent can take place by monitoring for a change in light emission fromthe signaling chromophore that occurs upon such hybridization during theamplification scheme.

The Polycationic Multichromophore

Light harvesting multichromophore systems have been demonstrated to beefficient light absorbers by virtue of the multiple chromophores theycomprise. Examples include, but are not limited to, conjugated polymers,aggregates of conjugated molecules, luminescent dyes attached tosaturated polymers, semiconductor quantum dots and dendritic structures.For example, each repeat unit on a conjugated polymer can be consideredas a contributing chromophore, quantum dots are made up of many atoms, asaturated polymer can be functionalized with many luminescent dyemolecules on side chains, and dendrimers can be synthesized containingmany covalently bonded individual chromophores. Attachment ofchromophore assemblies onto solid supports, such as polymer beads orsurfaces, can also be used for light harvesting.

Light harvesting multichromophore systems can efficiently transferenergy to nearby luminescent species (e.g., “signaling chromophores”).Mechanisms for energy transfer include, for example, resonant energytransfer (Förster (or fluorescence) resonance energy transfer, FRET),quantum charge exchange (Dexter energy transfer) and the like.Typically, however, these energy transfer mechanisms are relativelyshort range; that is, close proximity of the light harvestingmultichromophore system to the signaling chromophore is required forefficient energy transfer. Under conditions for efficient energytransfer, amplification of the emission from the signaling chromophoreoccurs when the number of individual chromophores in the lightharvesting multichromophore system is large; that is, the emission fromthe signaling chromophore is more intense when the incident light (the“pump light”) is at a wavelength which is absorbed by the lightharvesting multichromophore system than when the signaling chromophoreis directly excited by the pump light.

Conjugated polymers (CPs) are characterized by a delocalized electronicstructure and can be used as highly responsive optical reporters forchemical and biological targets.^(12,13) Because the effectiveconjugation length is substantially shorter than the length of thepolymer chain, the backbone contains a large number of conjugatedsegments in close proximity Thus, conjugated polymers are efficient forlight harvesting and enable optical amplification via Forster energytransfer.¹⁴

Spontaneous interpolymer complexation between cationic polyelectrolytesand DNA has been described and is largely the result of cooperativeelectrostatic forces.^(15,16,17) Hydrophobic interactions betweenaromatic polymer units and DNA bases were also recentlyrecognized.^(18,19) The free energy of polyelectrolyte/DNA interactionsis controlled by the structure of the participating species used inconjunction with solution variables such as pH, ionic strength, andtemperature.²⁰ The strength and specificity of these interactions hasrecently been coordinated to recognize the tertiary structure of plasmidDNA.²¹

The multichromophores used in the present invention are polycationic andcan interact with a sensor polynucleotide electrostatically. Anypolycationic multichromophore that can absorb light and transfer energyto a signaling chromophore on a sensor polynucleotide can be used in themethods described. Exemplary multichromophores which can be used includeconjugated polymers, saturated polymers or dendrimers incorporatingmultiple chromophores in any viable manner, and semiconductornanocrystals (SCNCs). The conjugated polymers, saturated polymers anddendrimers can be prepared to incorporate multiple cationic species orcan be derivatized to render them polycationic after synthesis;semiconductor nanocrystals can be rendered polycationic by addition ofcationic species to their surface.

In a preferred embodiment, a conjugated polymer is used as thepolycationic multichromophore. A specific example is shown in structure1 where the cationic water soluble conjugated polymer ispoly((9,9-bis(6′-N,N,N-trimethylammonium)-hexyl)-fluorene phenylene)with iodide counteranions (denoted in the following as polymer 1).²² Theparticular size of this polymer is not critical, so long as it is ableto absorb light and transfer energy to signaling chromophores broughtinto proximity. Typical values of “n” fall within the range of two toabout 100,000. This specific molecular structure is not critical; anywater soluble cationic conjugated polymer with adequate luminescencequantum efficiency can be used.

Water soluble conjugated oligomers can also be used as the polycationicmultichromophore. An example of such a water soluble, cationic,luminescent conjugated oligomer with iodide counterions is shown below(denoted herein as oligomer 2):

Although the smaller oligomer 2 does not display the large signalamplification characteristic of a high molecular weight polymer, suchsmaller molecules are useful to deconvolute structure propertyrelationships, which are difficult to determine with the inherentpolydispersity and batch-to-batch variations found in polymers. Further,in aqueous media oligomers such as 2 are more soluble than theirpolymeric counterparts, and hydrophobic interactions are expected to beless important for 2 than for polymer structures. Assemblies ofoligomers may thus be desirably used for specific applications. Additionof organic solvents, for example a water miscible organic solvent suchas ethanol, can result in a decrease in background C* emission. Thepresence of the organic solvent can decrease hydrophobic interactionsand reduce background C* emission.

The Sensor Polynucleotide

A sensor polynucleotide is provided that is anionic and is complementaryto the target polynucleotide to be assayed, and has a predeterminedsequence. The sensor polynucleotide can be branched, multimeric orcircular, but is typically linear, and can contain nonnatural bases. Thesensor polynucleotide can be prepared with any desired sequence ofbases. Chemical methods for attaching the signaling chromophore to thesensor polynucleotide are known in the art.²³ Specific sensorpolynucleotide structures, including structures conjugated tochromophores, can be custom-made using commercial sources or chemicallysynthesized.

The Signaling Chromophore

Chromophores useful in the inventions described herein include anysubstance which can absorb energy from a polycationic multichromophorein an appropriate solution and emit light. For multiplexed assays, aplurality of different signaling chromophores can be used withdetectably different emission spectra. The chromophore can be alumophore or a fluorophore. Typical fluorophores include fluorescentdyes, semiconductor nanocrystals, lanthanide chelates,polynucleotide-specific dyes and green fluorescent protein.

Exemplary fluorescent dyes include fluorescein, 6-FAM, rhodamine, TexasRed, tetramethylrhodamine, carboxyrhodamine, carboxyrhodamine 6G,carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow,coumarin, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy-Chrome, phycoerythrin,PerCP (peridinin chlorophyll-a Protein), PerCP-Cy5.5, JOE(6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein), NED, ROX(5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue,Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor® 350,Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546,Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647,Alexa Fluor® 660, Alexa Fluor® 680, 7-amino-4-methylcoumarin-3-aceticacid, BODIPY® FL, BODIPY® FL-Br₂, BODIPY® 530/550, BODIPY® 558/568,BODIPY® 564/570, BODIPY® 576/589, BODIPY® 581/591, BODIPY® 630/650,BODIPY® 650/665, BODIPY® R6G, BODIPY® TMR, BODIPY® TR, conjugatesthereof, and combinations thereof. Exemplary lanthanide chelates includeeuropium chelates, terbium chelates and samarium chelates.

A wide variety of fluorescent semiconductor nanocrystals (“SCNCs”) areknown in the art; methods of producing and utilizing semiconductornanocrystals are described in: PCT Publ. No. WO 99/26299 published May27, 1999, inventors Bawendi et al.; U.S. Pat. No. 5,990,479 issued Nov.23, 1999 to Weiss et al.; and Bruchez et al., Science 281:2013, 1998.Semiconductor nanocrystals can be obtained with very narrow emissionbands with well-defined peak emission wavelengths, allowing for a largenumber of different SCNCs to be used as signaling chromophores in thesame assay, optionally in combination with other non-SCNC types ofsignaling chromophores.

Exemplary polynucleotide-specific dyes include acridine orange, acridinehomodimer, actinomycin D, 7-aminoactinomycin D (7-AAD),9-amino-6-chloro-2-methoxyacridine (ACMA), BOBO™-1 iodide (462/481),BOBO™-3 iodide (570/602), BO-PRO™-1 iodide (462/481), BO-PRO™-3 iodide(575/599), 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI),4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI),4′,6-diamidino-2-phenylindole, dilactate (DAPI, dilactate),dihydroethidium (hydroethidine), dihydroethidium (hydroethidine),dihydroethidium (hydroethidine), ethidium bromide, ethidium diazidechloride, ethidium homodimer-1 (EthD-1), ethidium homodimer-2 (EthD-2),ethidium monoazide bromide (EMA), hexidium iodide, Hoechst 33258,Hoechst 33342, Hoechst 34580, Hoechst S769121, hydroxystilbamidine,methanesulfonate, JOJO™-1 iodide (529/545), JO-PRO™-1 iodide (530/546),LOLO™-1 iodide (565/579), LO-PRO™-1 iodide (567/580), NeuroTrace™435/455, NeuroTrace™ 500/525, NeuroTrace™ 515/535, NeuroTrace™ 530/615,NeuroTrace™ 640/660, OliGreen, PicoGreen® ssDNA, PicoGreen® dsDNA,POPO™-1 iodide (434/456), POPO™-3 iodide (534/570), PO-PRO™-1 iodide(435/455), PO-PRO™-3 iodide (539/567), propidium iodide, RiboGreen®,SlowFade®, SlowFade® Light, SYBR® Green I, SYBR® Green II, SYBR® Gold,SYBR® 101, SYBR® 102, SYBR® 103, SYBR® DX, TO-PRO®-1, TO-PRO®-3,TO-PRO®-5, TOTO®-1, TOTO®-3, YO-PRO®-1 (oxazole yellow), YO-PRO®-3,YOYO®-1, YOYO®-3, TO, SYTOX® Blue, SYTOX® Green, SYTOX® Orange, SYTO® 9,SYTO® BC, SYTO® 40, SYTO® 41, SYTO® 42, SYTO® 43, SYTO® 44, SYTO® 45,SYTO® Blue, SYTO® 11, SYTO® 12, SYTO® 13, SYTO® 14, SYTO® 15, SYTO® 16,SYTO® 20, SYTO® 21, SYTO® 22, SYTO® 23, SYTO® 24, SYTO® 25, SYTO® Green,SYTO® 80, SYTO® 81, SYTO® 82, SYTO® 83, SYTO® 84, SYTO® 85, SYTO®Orange, SYTO® 17, SYTO® 59, SYTO® 60, SYTO® 61, SYTO® 62, SYTO® 63,SYTO® 64, SYTO® Red, netropsin, distamycin, acridine orange,3,4-benzopyrene, thiazole orange, TOMEHE, daunomycin, acridine,pentyl-TOTAB, and butyl-TOTIN. Asymmetric cyanine dyes may be used asthe polynucleotide-specific dye. Other dyes of interest include thosedescribed by Geierstanger, B. H. and Wemmer, D. E., Annu. Rev. Vioshys.Biomol. Struct. 1995, 24, 463-493, by Larson, C. J. and Verdine, G. L.,Bioorganic Chemistry: Nucleic Acids, Hecht, S. M., Ed., OxfordUniversity Press: New York, 1996; pp 324-346, and by Glumoff, T. andGoldman, A. Nucleic Acids in Chemistry and Biology, 2^(nd) ed.,Blackburn, G. M. and Gait, M. J., Eds., Oxford University Press: Oxford,1996, pp 375-441. The polynucleotide-specific dye may be anintercalating dye, and may be specific for double-strandedpolynucleotides. Other dyes and fluorophores are described atwww.probes.com (Molecular Probes, Inc.).

The term “green fluorescent protein” refers to both native Aequoreagreen fluorescent protein and mutated versions that have been identifiedas exhibiting altered fluorescence characteristics, including alteredexcitation and emission maxima, as well as excitation and emissionspectra of different shapes (Delagrave, S. et al. (1995) Bio/Technology13:151-154; Heim, R. et al. (1994) Proc. Natl. Acad. Sci. USA91:12501-12504; Heim, R. et al. (1995) Nature 373:663-664). Delgrave etal. isolated mutants of cloned Aequorea victoria GFP that hadred-shifted excitation spectra. Bio/Technology 13:151-154 (1995). Heim,R. et al. reported a mutant (Tyr66 to His) having a blue fluorescence(Proc. Natl. Acad. Sci. (1994) USA 91:12501-12504).

In one variation, a second signaling chromophore, which may be directlyor indirectly attached to another of the assay components and/or to asubstrate, is used to receive energy from the initial signalingchromophore. In particular applications, this can provide forsignificant additional selectivity. For example, apolynucleotide-specific dye can be used as either the initial or secondsignaling chromophore, and may be specific for double-strandedsequences. Energy can then be transferred from the excited cationicmultichromophore to the initial signaling chromophore, whichsubsequently transfers energy to the second signaling chromophore, in anoverall format that is selective for the target. This cascade ofsignaling chromophores can, in principle, be extended to use any numberof signaling chromophores with compatible absorption and emissionprofiles. In one embodiment of this variation, an intercalating dye thatis specific for double-stranded polynucleotides is used as the secondsignaling chromophore, and an initial signaling chromophore that iscapable of transferring energy to the second signaling chromophore isconjugated to the sensor polynucleotide. The intercalating dye providesthe added selective requirement that the sensor and targetpolynucleotides hybridize before it is recruited to the detectioncomplex. In the presence of target, the duplex is formed, the dye isrecruited, and excitation of the multichromophore leads to signalingfrom the second signaling chromophore.

The Substrate

The methods described herein can be performed on a substrate in any of avariety of formats. The substrate can comprise a wide range of material,either biological, nonbiological, organic, inorganic, or a combinationof any of these. For example, the substrate may be a polymerizedLangmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO₂,SiN₄, modified silicon, or any one of a wide variety of gels or polymerssuch as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride,polystyrene, cross-linked polystyrene, polyacrylic, polylactic acid,polyglycolic acid, poly(lactide coglycolide), polyanhydrides,poly(methyl methacrylate), poly(ethylene-co-vinyl acetate),polysiloxanes, polymeric silica, latexes, dextran polymers, epoxies,polycarbonates, agarose, poly(acrylamide) or combinations thereof.Conducting polymers and photoconductive materials can be used.

Substrates can be planar crystalline substrates such as silica basedsubstrates (e.g. glass, quartz, or the like), or crystalline substratesused in, e.g., the semiconductor and microprocessor industries, such assilicon, gallium arsenide, indium doped GaN and the like, and includessemiconductor nanocrystals.

The substrate can take the form of a photodiode, an optoelectronicsensor such as an optoelectronic semiconductor chip or optoelectronicthin-film semiconductor, or a biochip. The location(s) of the individualsensor polynucleotide(s) on the substrate can be addressable; this canbe done in highly dense formats, and the location(s) can bemicroaddressable or nanoaddressable.

Silica aerogels can also be used as substrates, and can be prepared bymethods known in the art. Aerogel substrates may be used as freestanding substrates or as a surface coating for another substratematerial.

The substrate can take any form and typically is a plate, slide, bead,pellet, disk, particle, microparticle, nanoparticle, strand,precipitate, optionally porous gel, sheets, tube, sphere, container,capillary, pad, slice, film, chip, multiwell plate or dish, opticalfiber, etc. The substrate can be any form that is rigid or semi-rigid.The substrate may contain raised or depressed regions on which a sensorpolynucleotide or other assay component is located. The surface of thesubstrate can be etched using well known techniques to provide fordesired surface features, for example trenches, v-grooves, mesastructures, or the like.

Surfaces on the substrate can be composed of the same material as thesubstrate or can be made from a different material, and can be coupledto the substrate by chemical or physical means. Such coupled surfacesmay be composed of any of a wide variety of materials, for example,polymers, plastics, resins, polysaccharides, silica or silica-basedmaterials, carbon, metals, inorganic glasses, membranes, or any of theabove-listed substrate materials. The surface can be opticallytransparent and can have surface Si—OH functionalities, such as thosefound on silica surfaces.

The substrate and/or its optional surface are chosen to provideappropriate optical characteristics for the synthetic and/or detectionmethods used. The substrate and/or surface can be transparent to allowthe exposure of the substrate by light applied from multiple directions.The substrate and/or surface may be provided with reflective “mirror”structures to increase the recovery of light.

The substrate and/or its surface is generally resistant to, or istreated to resist, the conditions to which it is to be exposed in use,and can be optionally treated to remove any resistant material afterexposure to such conditions.

Sensor polynucleotides can be fabricated on or attached to the substrateby any suitable method, for example the methods described in U.S. Pat.No. 5,143,854, PCT Publ. No. WO 92/10092, U.S. patent application Ser.No. 07/624,120, filed Dec. 6, 1990 (now abandoned), Fodor et al.,Science, 251: 767-777 (1991), and PCT Publ. No. WO 90/15070). Techniquesfor the synthesis of these arrays using mechanical synthesis strategiesare described in, e.g., PCT Publication No. WO 93/09668 and U.S. Pat.No. 5,384,261.

Still further techniques include bead based techniques such as thosedescribed in PCT Appl. No. PCT/US93/04145 and pin based methods such asthose described in U.S. Pat. No. 5,288,514.

Additional flow channel or spotting methods applicable to attachment ofsensor polynucleotides to the substrate are described in U.S. patentapplication Ser. No. 07/980,523, filed Nov. 20, 1992, and U.S. Pat. No.5,384,261. Reagents are delivered to the substrate by either (1) flowingwithin a channel defined on predefined regions or (2) “spotting” onpredefined regions. A protective coating such as a hydrophilic orhydrophobic coating (depending upon the nature of the solvent) can beused over portions of the substrate to be protected, sometimes incombination with materials that facilitate wetting by the reactantsolution in other regions. In this manner, the flowing solutions arefurther prevented from passing outside of their designated flow paths.

Typical dispensers include a micropipette optionally roboticallycontrolled, an ink-jet printer, a series of tubes, a manifold, an arrayof pipettes, or the like so that various reagents can be delivered tothe reaction regions sequentially or simultaneously.

Excitation and Detection of the Chromophores

Any instrument that provides a wavelength that can excite thepolycationic multichromophore and is shorter than the emissionwavelength(s) to be detected can be used for excitation. The excitationsource preferably does not significantly excite the signalingchromophore directly. The source may be: a broadband UV light sourcesuch as a deuterium lamp with an appropriate filter, the output of awhite light source such as a xenon lamp or a deuterium lamp afterpassing through a monochromator to extract out the desired wavelengths,a continuous wave (cw) gas laser, a solid state diode laser, or any ofthe pulsed lasers. The emitted light from the signaling chromophore canbe detected through any suitable device or technique; many suitableapproaches are known in the art. For example, a fluorometer orspectrophotometer may be used to detect whether the test sample emitslight of a wavelength characteristic of the signaling chromophore uponexcitation of the multichromophore.

Kits

Kits comprising reagents useful for performing the methods of theinvention are also provided. In one embodiment, a kit comprises asingle-stranded sensor polynucleotide that is complementary to a targetpolynucleotide of interest and a polycationic multichromophore. Thesensor polynucleotide is conjugated to a signaling chromophore. In thepresence of the target polynucleotide in the sample, the sensorpolynucleotide hybridizes to the target, resulting in increased emissionof energy from the signaling chromophore, which can be detected.

The components of the kit are retained by a housing. Instructions forusing the kit to perform a method of the invention can be provided withthe housing, and can be provided in any fixed medium. The instructionsmay be located inside the housing or outside the housing, and may beprinted on the interior or exterior of any surface forming the housingwhich renders the instructions legible. The kit may be in multiplexform, containing pluralities of one or more different sensorpolynucleotides which can hybridize to corresponding different targetpolynucleotides.

EXAMPLES

The following examples are set forth so as to provide those of ordinaryskill in the art with a complete description of how to make and use thepresent invention, and are not intended to limit the scope of what isregarded as the invention. Efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperature, etc.) but someexperimental error and deviation should be accounted for. Unlessotherwise indicated, parts are parts by weight, temperature is degreecentigrade and pressure is at or near atmospheric, and all materials arecommercially available.

Example 1 Identification of a FRET Scheme

Hybridization tests using energy transfer from the light harvestingmulti-chromophore system to the signaling chromophore was demonstratedusing the cationic water soluble conjugated polymerpoly(9,9-bis(6′-N,N,N-trimethylammonium)-hexyl)-fluorene phenylene),polymer 1 with iodide counteranions. The sensor polynucleotide sequencewas 5′-GTAAATGGTGTTAGGGTTGC-3′ (SEQ ID NO: 1), corresponding to theanthrax (Bacillus anthracis) spore encapsulation plasmid, pX02, withfluorescein at the 5′ position, forming an example of oligo-C*.²⁴ Theabsorption and emission spectra of the polymer and the signalingchromophore are shown in FIG. 1. The data show an optical window for theexcitation of polymer 1, between the absorption of DNA and fluorescein.Direct excitation of polymer 1 results in energy transfer (ET) tofluorescein, as shown in FIG. 1. The absorbance overlap of fluoresceinwith the emission of polymer 1 was selected to ensure FRET.²⁵ The extentof ET can be evaluated by the ratio of integrated acceptor to donoremission.

Example 2 Demonstration of FRET in the Presence of Target Polynucleotide

Hybridization of the Oligo-C* probe leads to changes in the ET ratio.The sensor polynucleotide ([Oligo-C*]=2.1×10⁻⁸M) was annealed at 2° C.below its T_(m) (58.4° C.) in the presence of an equal molar amount of a40 base pair strand containing a complementary 20 base pair sequence,5′-CATCTGTAAATCCAAGAGTAGCAACCCTAACACCATTTAC-3′ (SEQ ID NO: 2), and in anidentical fashion with a non-complementary 40 base strand with thesequence 5′-AAAATATTGTGTATCAAAATGTAAATGGTGTTAGGGTTGC-3′ (SEQ ID NO: 3).Direct comparison of the resulting fluorescence reveals an ET ratiogreater than 6 fold higher for the hybridized DNA. See FIG. 2. It isalso highly significant that these optical differences are observed inthe presence of a 10 mmol Sodium Citrate and 100 mmol Sodium Chloridebuffer. Buffer ions screen like charges on complementary DNA strandswhich facilitates hybridization but weakens electrostatic interactionsbetween CPs and fluorescence quenchers of opposite charge.²⁶ Using aXenon lamp fluorometer, equipped with a standard PMT, the hybridized DNAprovided over 3 fold greater ET ratios, at [sensorpolynucleotide]=2.8×10⁻⁹M, than did the non-hybridized DNA.

Example 3 Optimization of Energy Transfer

Energy transfer was optimized by varying the ratio of polymer 1 toOligo-C*. At a concentration of [Oligo-C*]=2.1×10⁻⁸M, initial additionsof polymer cause an immediate rise in the ET ratio, which drops off asthe amount of polymer 1 begins to far exceed that of Oligo-C*. Themaximum in the ET ratio corresponds to a near 1:1 ratio of polymerchains to Oligo-C*. At high polymer concentrations not every chain is astightly complexed to Oligo-C* and the donor emission rises faster thanthat of the signaling chromophore. Such a relationship is expected,since when [polymer 1]/[Oligo-C*]<1, not all sensor polynucleotidestrands can be complexed efficiently to independent polymer chains.Conversely, in the [polymer 1]/[Oligo-C*]>1 regime, not all the photonsharnessed by polymer 1 (the donor) can be transferred to the Oligo-C*(the acceptor). Once the repeat unit of polymer to Oligo-C* reachesapproximately 100, the photoluminescence of the reporting chromophore nolonger increases, indicating acceptor saturation. Integratedfluorescence emission at this ratio was ˜4 fold greater than that of thedirectly excited (480 nm) probe in the absence of polymer 1, givingfurther evidence of signal amplification.

Example 4 FRET Transfer from a Multichromophore Through a SensorPolynucleotide to a Polynucleotide Dye for Improved Selectivity

Hybridization of the Oligo-C* probe (5′-Fl-ATCTTGACTATGTGGGTGCT-3′) (SEQID NO: 4) leads to differences in the ET ratio to two different sensorchromophores. The sensor polynucleotide ([Oligo-C*]=1×10⁻⁸M) wasannealed at 2° C. below its T_(m) (58.5° C.) in the presence of an equalmolar amount of a 20 base pair strand containing a complementary 20 basepair sequence, (5′-AGCACCCACATAGTCAAGAT-3′) (SEQ ID NO: 5), and in anidentical fashion with a non-complementary 20 base pair strand with thesequence (5′-CGTATCACTGGACTGATTGG-3′) (SEQ ID NO: 6). The two DNAmixtures were mixed with Ethidum Bromide ([EB]=1.1×10⁻⁶M) at roomtemperature in potassium phosphate monobasic-sodium hydroxide buffersolution (50 mM, pH=7.40) where the intercalation of the EB occurredwithin the duplex structure of the hybridized DNA pair. Addition ofpolymer 1 in water ([1]=1.6×10⁻⁷M) and subsequent excitation of 1 (380nm) resulted in energy transfer from 1 to the sensor polynucleotide andthen energy transfer from the sensor polynucleotide to EB. Comparison ofthe resulting fluorescence reveals that the sensor solution onlyexhibited emission from the intercalating dye EB in the presence of thehybridized DNA. See FIG. 3. This result demonstrates that the DNAsequence sensor of this invention can detect the presence of targetsingle stranded DNA with a specific base sequence complementary to thatof the sensor polynucleotide by detecting the emission from apolynucleotide-specific dye upon excitation of a polycationicmultichromophore. The chromophores used in this example were chosen forthe proper overlaps in energy between the multichromophore and the twosignaling chromophores.

Example 5 Demonstration of Optical Amplification of Polynucleotide DyesUsing FRET from Multichromophore Systems

Excitation of 1 in a solution of 1, sensor polynucleotide andpolynucleotide specific dye (Ethidium Bromide, EB) resulted in emissionintensities of EB that were ˜8 fold greater than that of the directlyexcited (500 nm) EB contained in the same double stranded DNA sequencewhich lacked the signaling chromophore on the sensor polynucleotide.Direct excitation of the signaling Oligo-C* (480 nm), in the absence ofpolymer 1, only provided an approximate 4 fold sensitization of theintercalated EB. Altogether, this example shows evidence of signalamplification by FRET from the polycationic multichromophore (conjugatedpolymer 1) to the diagnostic EB reporter. See FIG. 4.

Example 6 FRET Transfer to a Polynucleotide Dye

Experiments using 1 and ethidium bromide (“EB”) as a signalingchromophore demonstrated that direct energy transfer from 1 to EB couldbe shown in the presence of double-stranded DNA. The sensorpolynucleotide (5′-ATCTTGACTATGTGGGTGCT-3′) (SEQ ID NO: 4) lacking thesignaling chromophore ([Oligo]=1×10⁻⁸ M) was annealed at 2° C. below itsT_(m) (58.5° C.) in the presence of an equal molar amount of a 20 basepair strand containing a complementary 20 base pair sequence,(5′-AGCACCCACATAGTCAAGAT-3′) (SEQ ID NO: 5), and in an identical fashionwith a non-complementary 20 base pair strand with the sequence(5′-CGTATCACTGGACTGATTGG-3′) (SEQ ID NO: 6). The two DNA mixtures weremixed with Ethidum Bromide ([EB]=1.1×10⁻⁶ M) at room temperature inpotassium phosphate monobasic-sodium hydroxide buffer solution (50 mM,pH=7.40) where the intercalation of the EB occurred within the duplexstructure of the hybridized DNA pair. Addition of polymer 1 in water([1]=1.6×10⁻⁷ M) and subsequent excitation of 1 (380 nm) resulted inenergy transfer from 1 to the intercalated EB only in the case ofhybridized or double stranded DNA. Emission from the EB was detectedupon excitation of polymer 1 for only the hybridized sequences. See FIG.5.

Although the invention has been described in some detail with referenceto the preferred embodiments, those of skill in the art will realize, inlight of the teachings herein, that certain changes and modificationscan be made without departing from the spirit and scope of theinvention. Accordingly, the invention is limited only by the claims.

REFERENCES

-   ¹ Wang, J. Nucleic Acid Res. 2000 28 3011.-   ² Umek, R. M.; Lin, S. W.; Vielmetter, J.; Terbrueggen, R. H.;    Irvine, B.; Yu, C. J.; Kayyem, J. F.; Yowanto, H.; Blackburn, G. F.;    Farkas, D. H.; Chen, Y. P. J. Mol. Diag. 2001 3 74.-   ³ Schork N. J.; Fallin D.; Lanchbury J. S. Clini. Genet. 2000 58    250.-   ⁴ Balakin, K. V.; Korshun, V. A.; Mikhalev, I. I.; Maleev, G. V.;    Malakhov A. D.; Prokhorenko, I. A.; Berlin, Yu. A. Biosensors and    Bioelectronics 1998 13 771.-   ⁵ LePecg, J. B.; Paoletti, C. J. Mol. Biol. 1967 27 87.-   ⁶ Petty, J. T.; Bordelon, J. A.; Robertson, M. E. J. Phys. Chem. B    2000 104 7221.-   ⁷ Cardullo, R. A.; Agrawal, S.; Flores, C.; Zamechnik, P. C.;    Wolf, D. E. Proc. Natl. Acad. Sci. 1988 85 8790.-   ⁸ Castro, A.; Williams, J. G. K. Anal. Chem. 1997 69 3915.-   ⁹ Knemeyer, J.; Marmé, N.; Sauer, M. Anal. Chem. 2000 72 3717.-   ¹⁰ Minehan, D. S.; Marx, D. A.; Tripathy, S. K. Macromolecules 1994    27 777.-   ¹¹ Pullman, B.; Lavery, R.; Pullman, A. Eur. J. Biochem. 1982 124    229.-   ¹² McQuade, D. T.; Pullen. A. E.; Swager, T. M. Chem. Rev. 2000 100    2537.-   ¹³ Chen, L.; McBranch, D. W.; Wang, H.-L.; Helgeson, R.; Wudl, F.;    Whitten, D. G. Proc. Natl. Acad. Sci. U.S.A. 1999 96 12287.-   ¹⁴ Dogariu, A., Gupta, R., Heeger, A. J., Wang, H. Synthetic Metals    1999 100 95.-   ¹⁵ Kabanov, A. V.; Felgner, P.; Seymour, L. W., Eds. Self-Assembling    Complexes for Gene Delivery. From Laboratory to Clinical Trial; John    Wiley: Chichester, 1998.-   ¹⁶ Kircheis, R.; Blessing, T.; Brunner, S.; Wightman, L.;    Wagner, E. J. Controlled Release 2001 72 165.-   ¹⁷ Wolfert, M. A.; Dash, P. R.; Navarova, O.; Oupicky, D.;    Seymour, L. W.; Smart, S.; Strohalm, J.; Ulbrich, K. Bioconjugate    Chem. 1999 10 993.-   ¹⁸ Ganachaud, F.; Elaïssari, A.; Pichot, C.; Laayoun, A.; Cros, P.    Langmuir 1997 13 701.-   ¹⁹ Smith, J. O.; Olson, D. A.; Armitage, B. A. J. Am. Chem, Soc.    1999 121 2628.-   ²⁰ Harada, A.; Kataoka, K. Science 1999 283 65.-   ²¹ Bronich, T. K.; Nguyen, H. K.; Eisenberg, A.; Kabanov, A. V. J.    Am. Chem. Soc. 2000 122 8339.-   ²² Stork, M. S.; Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. Adv.    Mater. 2002 14 361. The molecular weight of polymer 1 was determined    to be 8,600 g/mole (Mn).-   ²³ Nielsen, P. E.; Egholm, M. Peptide Nucleic Acids: Protocols and    Applications. Horizon Scientific Press, Portland, 1999.-   ²⁴ Makino, S.-I.; Uchida, I.; Terakado, N.; Sasakawa, C.; Yoshikawa,    M.; J. Bacteriol. 1989, 171, 722.-   ²⁵ Lakowicz, J. R. Principles of Fluorescence Spectroscopy. Kluwer    Academic/Plenum Publishers: New York, 1999.-   ²⁶ Wang, J.; Wang, D. L.; Miller, E. K.; Moses, D.; Bazan, G. C.;    Heeger, A. J. Macromolecules 2000, 33, 5153.

1. An assay method for detecting polynucleotide binding comprising: a.providing a sample that is suspected of containing a firstpolynucleotide; b. providing a second polynucleotide that iscomplementary to the first polynucleotide; c. providing a signalingchromophore; d. providing a conjugated polymer that upon excitation iscapable of transferring energy to the signaling chromophore; e.contacting the sample with the second polynucleotide, the signalingchromophore and the conjugated polymer under conditions in which thefirst and second polynucleotides can hybridize to form a signalingcomplex further comprising the conjugated polymer and the signalingchromophore; and f. determining if the first and second polynucleotideshave hybridized by detecting if light is produced from the signalingchromophore upon excitation of the conjugated polymer.
 2. The method ofclaim 1, wherein the amount of emitted light produced from the signalingchromophore upon excitation of the signaling complex is greater thanthat obtained from direct excitation of the signaling chromophore. 3.The method of claim 1, wherein the conjugated polymer has the structure


4. The method of claim 1, wherein the conjugated polymer is an aggregateof conjugated molecules.
 5. The method of claim 1, wherein the sample iscontacted with the second polynucleotide and the conjugated polymer inthe presence of a sufficient amount of an organic solvent to decreasehydrophobic interactions with the conjugated polymer.
 6. The method ofclaim 1, wherein the method is performed in a multiplex format.
 7. Themethod of claim 6, wherein the sample is contacted with a plurality ofdifferent second polynucleotides having corresponding differentsequences, wherein each of said different second polynucleotides canselectively hybridize to a corresponding different complementary firstpolynucleotide resulting in different signaling complexes, wherein eachof said different signaling complexes comprises a correspondingdifferent signaling chromophore.
 8. The method of claim 1, wherein thesignaling chromophore is a fluorophore.
 9. The method of claim 7,wherein the fluorophore is selected from a semiconductor nanocrystal, afluorescent dye, a green fluorescent protein, and a lanthanide chelate,and a polynucleotide-specific dye.
 10. The method of claim 8, whereinthe fluorophore is a fluorescent dye.
 11. The method of claim 1, whereinthe amount of light emitted from the signaling chromophore isquantitated and used to determine the amount of a polynucleotide in thesample.
 12. The method of claim 1, wherein the method is performed on asubstrate.
 13. The method of claim 12, wherein the substrate is selectedfrom the group consisting of a bead, a chip, a microsphere, a slide, amultiwell plate, an optical fiber, an optionally porous gel matrix, aphotodiode, and an optoelectronic device.
 14. The method of claim 13,wherein the substrate is a bead.
 15. The method of claim 13, wherein thesubstrate is a slide and detecting polynucleotide binding comprisesperforming fluorescence in situ hybridization.
 16. The method of claim12, wherein the substrate is nanoaddressable or microaddressable. 17.The method of claim 12, wherein the substrate is conjugated to aplurality of different second polynucleotides having correspondingdifferent sequences, wherein each of said different sensorpolynucleotides can selectively hybridize to a corresponding differentfirst polynucleotide to form a signaling complex with the conjugatedpolymer.
 18. A polynucleotide sensing solution comprising: a signalingchromophore; and a conjugated polymer that is capable of transferringenergy to the signaling chromophore upon excitation when in proximitythereto, wherein a greater amount of energy is emitted from thesignaling chromophore upon excitation of the conjugated polymer in thepresence of the polynucleotide being sensed.
 19. A method of performingfluorescence energy transfer comprising: a. adding a conjugated polymer,a sensor polynucleotide and a signaling chromophore to a biologicalsample; and b. detecting emission from the signaling chromophore byexciting the conjugated polymer, wherein the emission from the signalingchromophore is greater than that obtained from direct excitation. 20.The method of claim 19, wherein the emission is at least 4 times largerthan that obtained from direct excitation of the signaling chromophore.21. A composition comprising: a. a conjugated polymer, and b. asignaling chromophore, wherein the conjugated polymer when excitedtransfers energy to the signaling chromophore, upon which the signalingchromophore emits a signal at least four-fold greater than when directlyexcited.
 22. A signaling complex formed by the method of claim 1.